Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Disclosed are a cancer marker-detecting composition comprising an agent
for measuring an mRNA or protein expression level of eIF3m, a cancer
diagnosis kit comprising the same, a method for detecting an eIF3m
polynucleotide or protein by treating a biological specimen with the
agent to detect a substance binding specifically to the agent and
quantitatively comparing the substance between a subject and a normal
control, and a method for the treatment and prevention of cancer
comprising an agent for down-regulating the expression of an eIF3m
polynucleotide or protein.

Claims:

1. A composition for detecting a cancer marker, comprising an agent for
measuring an mRNA or protein expression level of eIF3m.

2. The composition according to claim 1, wherein the agent for measuring
an mRNA expression level of eIF3m specifically binds to the eIF3m mRNA
and is selected from among primer pairs, probes, and antisense
oligonucleotides.

3. The composition according to claim 1, wherein the agent for measuring
a protein expression level of eIF3m is an antibody.

4. The composition according to claim 1, wherein the cancer marker is
targeted for cancer selected from a group consisting of lung cancer,
breast cancer, liver cancer, leukemia, lymphoma, colon cancer, melanoma
and rectal cancer.

5. A cancer diagnosis kit, comprising the composition of one of claims 1
to 4.

6. The cancer diagnosis kit according to claim 5, being based on an
RT-PCR kit, a competitive RT-PCR kit, a real-time RT-PCR kit, a DNA chip
kit or a protein chip kit.

7. A method for detecting an eIF3m polynucleotide, comprising: (a)
providing a biological specimen; (b) treating the biological specimen
with the agent of claim 2; (c) detecting a conjugate of the agent with a
polynucleotide complementary thereto; and (d) quantitatively comparing
the conjugate between a subject and a normal control.

8. A method for detecting an eIF3m protein, comprising: (a) providing a
biological specimen; (b) treating the biological specimen with an
antibody specific for the eIF3m protein; (c) detecting an
antigen-antibody complex formed; and (d) quantitatively comparing the
complex between a subject and a normal control.

9. A composition for treatment and prevention of cancer, comprising an
oligonucleotide inhibitory of expression of an eIF3m polynucleotide.

10. The composition according to claim 9, wherein the oligonucleotide is
an antisense oligonucleotide, an aptamer, siRNA or shRNA, which is
specific for an eIF3m gene.

11. A composition for treatment and prevention of cancer, comprising an
antibody having inhibitory activity against an eIF3m polypeptide or a
antigen-binding fragment thereof.

12. The composition according to claim 9 or 11, wherein the cancer is
selected from a group consisting of lung cancer, breast cancer, leukemia,
lymphoma, colon cancer, melanoma and rectal cancer.

13. A method for screening a curative drug for cancer, comprising:
treating a cell expressing an eIF3m polypeptide and/or polynucleotide
with a candidate compound; and measuring an eIF3m polypeptide or
polynucleotide expression level in the cell.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a cancer diagnosis composition
comprising an agent for measuring an expression level of eIF3m and a
composition for the treatment and prevention of cancer by regulating the
expression level. More particularly, the present invention pertains to a
cancer marker-detecting composition comprising an agent for measuring an
mRNA or protein expression level of eIF3m, a cancer diagnosis kit
comprising the same, a method for detecting an eIF3m polynucleotide or
protein by treating a biological specimen with the agent to detect a
substance binding specifically to the agent and quantitatively comparing
the substance between a subject and a normal control, and a method for
the treatment and prevention of cancer comprising an agent for
down-regulating the expression of an eIF3m polynucleotide or protein.

BACKGROUND ART

[0002] Cancer is one of the most common causes of morbidity and mortality
throughout the world today. Cancer incidence is expected to increase due
to increasing average life expectancy, with the onset age being lowered.
The ACS (American Cancer Society)'s annual Cancer Statistics article
reports that in 2007, 12 million or more new cancer cases were diagnosed
worldwide, with the death toll of about 7.6 million cancer patients at a
death rate of about twenty thousands per day.

[0003] Lung cancer, breast cancer and colon cancer are representative of
the most deadly cancer. Particularly with regard to colon cancer, its
incidence of colon cancer has been dramatically increasing in South
Korea. It is the fourth leading cause of cancer-related death among men
in Korea, after stomach cancer, lung cancer and liver cancer. Similar
rates of cancer mortality are found for women. Most cases occur among
patients in their 50s, and secondly, in the 60s. The age of the greatest
incidence of colon cancer in Korea is 10 years lower than that in the
Western world in countries such as the U.S.A. and the Europe. In the 30s,
the high incidence frequency of colon cancer accounts for 5% - 10% of all
cases. Cases in the young are uncommon unless a family history of early
colon cancer is present. Factors which have an influence on
carcinogenesis are, for the most part, environmental, such as the
westernization of the diet, particularly excess intake of animal fats and
proteins, rather than heredity. Only 5% of colon cancer cases are
attributed to hereditary predisposition. According to this fact and
recent reports, persons with a high risk of developing colon cancer are
those who 1) have been affected by colon polyps, 2) have a family history
of colon cancer, 3) suffer from ulcerative colitis for a long period of
time, or 4) are attacked by intractable anal fistula.

[0004] When detected at an early stage, colon cancer can be almost
completely cured by endoscopic resection or surgical operation. Further,
although metastasized to the liver or the lung (distant metastasis),
colon cancer may still be completely cured through surgical therapy
unless found too late to be operated upon. It is, however, very difficult
to detect colon cancer in asymptomatic patients since the patients with
colon cancer have no subjective symptoms in the early stage. In spite of
inconvenience and pain, accordingly, a periodic examination must be made
to detect colon cancer at the early stage which allows the surgical
operation to be applied for a cure. An occult blood test is considered to
be relatively convenient colon cancer screening. However, a positive
response in this test is often determined to be false positive. Likewise,
all negative responses do not guarantee the absence of colon cancer. That
is, it is unreasonable to use the occult blood test as an accurate
diagnostic method.

[0005] Leading to the present invention, intensive and thorough research
into the diagnosis of and therapeutics for colon cancer, resulted in the
finding that certain genes and their expression products can be used as
diagnostic markers for accurately detecting colon cancer in an early
stage and as targets for the treatment of colon cancer.

DISCLOSURE OF INVENTION

Technical Problem

[0006] Accordingly, it is an object of the present invention to provide a
composition for detecting a cancer marker, comprising an agent for
measuring an mRNA or protein ex- pression level of eIF3m.

[0007] It is another object of the present invention to provide a cancer
diagnosis kit, comprising an agent for measuring an mRNA or protein
expression level of eIF3m.

[0008] It is a further object of the present invention to provide a method
for detecting an eIF3m polynucleotide or protein, comprising treating a
biological specimen with the agent for measuring an mRNA or protein
expression level of eIF3m, detecting a complex of the agent with a
polynucleotide or protein complementary thereto, and quantitatively
comparing the complex between a subject and a normal control.

[0009] It is still a further object of the present invention to provide a
composition for the treatment and prevention of cancer, comprising an
oligonucleotide inhibitory of the expression of an eIF3m polynucleotide.

[0010] It is still another object of the present invention to provide a
composition for the treatment and prevention of cancer, comprising an
antibody having inhibitory activity against eIF3m polypeptide or the
antigen-binding fragment thereof.

[0011] It is still yet another object of the present invention to provide
a method for screening a curative drug for cancer, comprising treating a
cell expressing an eIF3m polypeptide and/or polynucleotide with a
candidate compound and measuring an eIF3m polypeptide or polynucleotide
expression level in the cell.

Solution to Problem

[0012] In accordance with an aspect thereof, the present invention
pertains to a composition for detecting a cancer marker, comprising an
agent for measuring an mRNA or protein expression level of eIF3m.

[0013] The term "eIF3m", as used herein, stands for eukaryotic translation
factor 3 m-subunit. eIF3 is a mammalian initiation factor with a
molecular weight of as large as ˜800 kDa. E1F3 is composed of 13
non-identical subunits designated eIF3a, b, c, . . . , m. Some of the
subunits are known to show aberrant expression in certain cancers, but
nowhere has information on the specific expression of the subunit of the
present invention in certain cancers been reported in previous documents,
thus far. eIF3m, also called PCID1 (PCI domain containing protein 1), was
known to act as a receptor or coreceptor for entry of herpes simplex
virus (HSV), but there have been no reports on the implication of the
subunit in tumorigenesis and further in therapy for cancer. Moreover, in
the present invention, eIF3m is first disclosed to be overexpressed in
specific cancerous cell lines and cancers. The present inventors
confirmed that eIF3m expression at both transcription and translation
levels drastically increases in human tumor tissues as well as in human
cancer cell lines. In the present invention, it is also found that eIF3m
is expressed at high levels in lung cancer, breast cancer, liver cancer,
leukemia, lymphoma, colon cancer, melanoma, and rectal cancer and that
elevated expression levels of eIF3m are maintained in tumor regions of
human colon tissues.

[0014] As used herein, the terms "marker" or "diagnosis marker" is
intended to indicate a substance capable of diagnosing cancer by
distinguishing cancer cells or subject suffering from cancer from normal
cells or subjects, and includes organic biological molecules, quantities
of which are increased or decreased in cancer cells relative to normal
cells, such as polypeptides, proteins or nucleic acids (e. g., mRNA,
etc.), lipids, glycolipids, glycoproteins and sugars (monosaccharides ,
disaccharides , oligosaccharides, etc.). With respect to the objects of
the present invention, the diagnosis marker of cancer is an eIF3m
polypeptide or a polynucleotide coding therefore, which are specifically
expressed at high levels in cancer cells, relative to normal cells or
tissues.

[0015] The term "measurement of mRNA expression levels" or corresponding
phrases, as used herein, are intended to refer to a process of assessing
the presence and expression levels of mRNA of cancer marker genes in
biological samples to diagnose cancer, in which the amount of mRNA is
measured. Analysis methods for measuring mRNA levels include, but are not
limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase
protection assay (RPA), Northern blotting and DNA chip assay.

[0016] The term "measurement of protein expression levels" or
corresponding phrases, as used herein, are intended to refer to a process
of assessing the presence and expression levels of proteins expressed
from colon cancer marker genes in biological samples to diagnose cancer,
in which the amount of protein products of the marker genes is measured
using antibodies specifically binding to the proteins. Analysis methods
for measuring protein levels include, but are not limited to, Western
blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay
(RIA) , radioimmunodiffusion, Ouchterlony immunodiffusion, rocket
immunoelectrophoresis, immunohistostaining, immunoprecipitation assay,
complement fixation assay, FACS(fluorescence activated cell sorter), and
protein chip assay.

[0017] The agent for measuring mRNA levels may be exemplified by a pair of
primers, probes, or antisense nucleotides, which are relevant to the
eIF3m polynucleotide or its fragments according to the present invention.
The primers, probes, or antisense nucleotide sequences may be easily
designed by those skilled in the art depending on the polynucleotide
sequence of the present invention.

[0018] As used herein, the term "primer" refers to a short nucleic acid
strand having a free 3' hydroxyl group, which forms a base pair with a
complementary template so as to serve as a starting point for the
production of a new template strand. DNA synthesis or replication
requires a suitable buffer, proper temperatures, polymerizing enzyme (DNA
polymerase, or reverse transcriptase), and four kinds of nucleotide
triphosphates, in addition to primers. In the present invention, sense
and antisense primers specific for the eIF3m polynucleotide can be used
for PCR amplification so as to diagnose cancer with the PCR products. The
sense and antisense primers may be altered in length suitably depending
on the information known in the art.

[0019] The term "probe", as used herein, is intended to refer to a
fragment of a nucleotide sequence, such as RNA or DNA, ranging in length
from as short as ones bases to as long as hundreds bases, which can bind
specifically to a mRNA of interest and which is tagged with a label for
detecting the mRNA of interest. The probe useful in the present invention
may be constructed in the form of oligonucleotide probes, single-stranded
DNA probes, double-stranded DNA probes, or RNA probes. In an embodiment
of the present invention, the diagnosis of cancer occurrence may be
achieved by determining whether a probe complementary to the eIF3m
polynucleotide of the present invention hybridizes with a nucleotide
sequence of interest. Selection of suitable probes and hybridization
conditions may be modified according to information known in the art.

[0020] The primers or probes useful in the present invention may be
chemically synthesized using a phosphoamidite solid support method or
other well-known techniques. Their nucleotide sequences may be modified
using various means known in the art. Illustrative, non-limiting examples
of the modification include methylation, capping, substitution of natural
nucleotides with one or more homologues, and alternation between
nucleotides, such as uncharged linkers (e.g., methyl phosphonate,
phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers
(e.g., phosphorothioate, phosphorodithioate, etc.).

[0021] Preferably, the primer or probe preferably contains 8 or more
nucleotides. Hybridization may be achieved by exposing or contacting the
primer or probe to the eIF3m polynucleotide of the present invention.
Preferably, these sequences are hybridized with each other under such a
stringent condition as to minimize non-specific pairings. In order to
detect sequences which share 80% to 90% homology with the eIF3m
polynucleotide of the present invention, for example, a hybridization
condition may include hybridizing overnight at 42° C. in a buffer
containing 0.25 M Na2HPO4, pH 7.2, 6.5% SDS, and 10% dextran
sulfate and finally washing at 50° C. with a solution containing
0.1×SSC and 0.1% SDS. A stringent condition suitable for detecting
a sequence which shares 90% homology with the eIF3m polynucleotide of the
present invention comprises hybridizing overnight at 65° C. in
0.25M Na2HPO4, pH7.2, 6.5% SDS, 10% dextran sulfate, and
finally washing at 60° C. with a solution containing 0.1×SSC
and 0.1% SDS.

[0022] In accordance with an embodiment of the present invention, the
agent for measuring the expression level of eIF3m protein (hereinafter,
used interchangeably with "eIF3m polypeptide") is preferably an antibody.

[0023] The term "antibody", as used herein, refers to a specific protein
molecule that indicates an antigenic region. With respect to the objects
of the present invention, the antibody binds specifically to the marker
of the present invention, that is, an eIF3m polypeptide. This antibody
can be produced from a protein which the marker gene cloned typically
into an expression vector encodes, using a conventional method. Also,
partial peptides producible from the protein encoded by the marker gene
fall within the scope of the antibody. For functioning as an antibody,
the partial peptide is required to contain at least 7 amino acid
residues, preferably 9 or more amino acid residues, and more preferably
12 or more amino acid residues. No particular limitations are imparted to
the form of the antibodies of the present invention. Among them are
polyclonal antibodies, monoclonal antibodies and fragments thereof which
contain a paratope, and all immunoglobulin antibodies. Further, special
antibodies such as humanized antibodies are also within the scope of the
present invention. Consequently, as long as it may be produced using a
method known in the art, any antibody against the eIF3m protein of the
present invention can be used in the present invention.

[0024] In addition, the antibodies of the present invention which are used
for detecting the diagnosis marker of cancer include functional fragments
of antibody molecules as well as complete forms having two full-length
light chains and two full-length heavy chains. The functional fragments
of antibody molecules refer to fragments retaining at least an
antigen-binding function, and include Fab, F(ab'), F(ab')2, Fv and the
like.

[0025] As used herein, the term "cancer" refers to a class of diseases in
connection with the regulation of cell death, in which a group of cells
display uncontrolled overgrowth, resulting from insufficient apopotosis.
The excessively growing cells invade adjacent tissues and organs to
destroy and deform normal structures, forming tumoral mass, the state of
which is defined as cancer. As a rule, tumor is a neoplasm or a solid
lesion formed by an abnormal excessive growth of cells. A tumor may be
benign or malignant. Malignant tumors, which typically grow far faster
than do benign tumors, invade adjacent tissues and sometimes metastasize,
threatening the life. The malignant tumor is typically regarded as
cancer. Examples of the cancers detectable with the composition for
detecting a cancer marker in accordance with the present invention
include cephaloma, head and neck cancer, lung cancer, breast cancer,
thymoma, mesothelioma, esophalgeal cancer, pancreatic cancer, colon
cancer, liver cancer, stomach cancer, cholangiocarcinoma, kidney cancer,
bladder, prostate cancer, testicular cancer, spermocytoma, ovarian
cancer, uterine cervical cancer, endometrial cancer, lymphoma, acute
leukemia, chromic leukemia, multiple myeloma, sarcoma, and malignant
melanoma, but are not limited thereto. In a preferred embodiment of the
present invention, the composition for detecting a cancer marker is
applied to lung cancer, liver cancer, colon cancer, breast cancer,
leukemia, lymphoma and melanoma cell lines to examine the expression
level of the cancer marker therein. When the composition was applied, the
eIF3m protein was observed to have remarkably higher expression levels in
tissues from subjects with cancer than in those from normal subjects.

[0026] In accordance with another aspect thereof, the present invention
provides a cancer diagnosis kit that comprises an agent for measuring an
mRNA or protein expression level of eIF3m.

[0027] The term "diagnosis" in the context of the present invention,
refers to a process of determining the presence or absence of the eIF3m
polypeptide or polynucleotide of the present invention in a biological
specimen or a tissue sample so as to identify the existence or
characteristics of a disease related to the expression of the gene.

[0028] The detection of cancer marker may be accomplished by determining
the expression level of the eIF3m polypeptide or a polynucleotide
encoding it using the kit of the present invention. The kit of the
present invention may comprise a primer or probe for measuring the
expression level of the cancer diagnosis marker, an antibody selectively
recognizing the cancer marker or its fragments retaining an
antigen-binding function, and/or one or more agents or compositions
suitable for the analysis of the polypeptide or polynucleotide. For
example, the diagnosis kit for the quantitative analysis of the
polynucleotide or gene of the present invention may comprise at least one
oligonucleotide specifically binding to a polynucleotide coding for the
eIF3m polypeptide. In a preferable embodiment, the diagnosis kit of the
present invention is characterized by including essential elements
required for performing RT-PCR. An RT-PCR kit includes a pair of primers
specific for the nucleotide sequence of eIF3m or its partial sequence,
reverse transcriptase, Taq polymerase, PCR primers, and dNTP. As long as
it takes advantage of analysis methods known in the context of
`measurement of mRNA expression level` any kit may be employed without
limitations.

[0029] In another preferable embodiment, the cancer diagnosis kit of the
present invention may comprise an antibody specifically binding to the
eIF3m protein of the present invention. As long as it takes advantage of
analysis methods known in the context of `measurement of protein
expression level`, any kit may be employed without limitations.
Preferable is an ELISA kit or a protein chip kit.

[0030] The measurement of protein expression level using an antibody is
based on the formation of an antigen-antibody complex between the eIF3m
protein and an antibody thereto. Leading to determining the protein
expression level, the amount of the antigen-antibody can be measured
using various methods.

[0031] As used herein, the term "antigen-antibody complex" is intended to
refer to binding products of a cancer marker protein to an antibody
specific thereto. The antigen-antibody complex thus formed may be
quantitatively determined by measuring the signal size of a detection
label.

[0032] For instance, cancer can be diagnosed by determining a significant
increase in eIF3m protein expression level in a suspected subject from
the comparison of the amount of antibody-antigen complex between the
suspected subject and a normal control. In this regard, a sample from a
subject with suspected cancer is treated with an antibody specific for
the eIF3m protein of the present invention to form an antigen-antibody
complex which can be quantitatively analyzed using a kit on the basis of
an ELISA assay, an RIA assay, a sandwich ELISA assay, a Western blotting
assay, a radioimmunodiffusion assay, an ouchterlony immunodiffusion
assay, a rocket immunoelectrophoresis assay, an immunohistostaining
assay, an immunoprecipitation assay, a complement fixation assay, FACS, a
protein chip assay or an immunodot assay. Comparison of the analysis data
with those of normal subject allows the diagnosis of cancer in connection
with an increase in eIF3m protein expression.

[0033] In accordance with a further aspect thereof, the present invention
pertains to a method for detecting an eIF3m polynucleotide or protein,
comprising treating a biological specimen with the agent for measuring an
mRNA or protein expression level of eIF3m, detecting a complex of the
agent with a polynucleotide or protein complementary thereto, and
quantitatively comparing the complex between a subject and a normal
control.

[0034] In detail, the expression of a gene may be detected at an mRNA or a
protein level. mRNA or protein isolation from a biological specimen may
be achieved using a well-known method.

[0035] As used therein, the term "biological specimen" is intended to
refer to a sample from which a gene or protein expression level of eIF3m
can be measured. Examples of the biological specimen useful in the
present invention include tissues, cells, whole blood, serum, plasma,
saliva, sputum, cerebrospinal fluid and urine, but are not limited
thereto.

[0036] In an embodiment of the detecting method according to the present
invention, a gene expression level in a subject with suspected cancer can
be compared to that in a normal control to diagnose cancer incidence in
the subject. In detail, a biological sample from a subject with suspected
cancer is measured for the expression level of the marker of the present
invention. This level is compared with that measured in a biological
sample from a normal control. When the expression level of the marker of
the present invention is higher in the subject than in the normal
control, the subject may be determined to be affected by cancer.

[0037] In the case where a polynuceotide coding for the eIF3m polypeptide
of the present invention is used as a marker, the method comprises (a)
providing a biological specimen; (b) treating the biological specimen
with an agent for measuring an expression level of eIF3m; (c) detecting a
binding product of the agent to a polynucleotide complementary to the
agent; and (d) quantitatively comparing the binding product between a
subject and a normal control. In the case where the eIF3m polypeptide of
the present invention is used as a marker, the method comprises (a)
providing a biological specimen; (b) treating the biological specimen
with an antibody specific for the eIF3m protein; (c) detecting an
antigen-antibody complex; and (d) quantitatively comparing the complex
between a subject and a normal control.

[0038] In accordance with still a further aspect thereof, the present
invention pertains to a composition for the treatment and prevention of
cancer, comprising as an active ingredient an oligonucleotide inhibitory
of the expression of an eIF3m polynucleotide or an antibody inhibiting
the activity of eIF3m polypeptide or an antigen-binding fragment thereof.

[0039] In a preferred embodiment of this aspect the pharmaceutical
composition may include a substance inhibiting the expression of eIF3m
polynucleotide of the present invention. The eIF3m expression inhibitor
substance may be selected from the group consisting of siRNA, shRNA, an
aptamer and an antisense oligonucleotide.

[0040] As used herein, the term "siRNA (small interfering RNA)" is
intended to refer to a small nucleic acid molecule of about 20
nucleotides, which mediates RNA interference or gene silencing. When
siRNA is introduced into a cell, it is recognized by dicer to degrade the
gene encoding the eIF3m, resulting in the specific knockdown of an eIF3m
gene.

[0041] The term "shRNA" refers to a short hairpin RNA in which sense and
antisense sequences of an siRNA target sequence are separated by a loop
structure of 5 to 9 bases.

[0042] As used herein, the term "aptamer" refers to an oligoribonucleic
acid molecule which is 20 to 60 nt long. It has various three-dimensional
structures depending on sequences and binds to a specific target molecule
to effectively regulate the function of the target molecule.

[0043] Recently, the phenomenon of RNA interference (RNAi) has been
studied for application to a method for controlling protein expression at
the gene level. Typically, siRNA has been shown to inhibit protein
expression by binding specifically to mRNA, having a sequence
complementary to a target gene.

[0044] In order to interfere with the expression of oncogenes or
metastagenes, the composition comprising siRNA or shRNA according to the
present invention may be administered to a subject according to a typical
method adopted for use in the gene therapy based on these RNAs. For
instance, gene expression can be regulated by low-volume intravenous
injection of siRNAs according to the method described by Filleur et al.,
Cancer Res., 63(14): 3919-22, 2003. In order to increase the cellular
uptake and stability of siRNAs, siRNA may also be injected in conjunction
with a conjugate according to Chien et al., Cancer Gene Ther., 12(3)
321-8, 2005.

[0045] The short interfering RNA molecules (siRNA) contained in the
present composition can be prepared by direct chemical synthesis (Sui G
et. al, (2002) Proc Natl Acad Sci USA 99:5515-5520) or in vitro
transcription (BrummelkampTR et al., (2002) Science 296:550-553), but the
present invention is not limited to these methods. Also, shRNAs, which
are designed to overcome the drawbacks of siRNAs, including expensive
siRNA biosynthesis and low transfection efficiency, leading to the
short-term persistence of the RNA interference effect, can be expressed
from a RNA polymerase III-based promoter contained in an adenoviral,
rentiviral or plasmid expression vector system, that has been introduced
into cells. The shRNA molecules are processed to functional siRNA
molecules using an siRNA processing enzyme (Dicer or RNase III) within
the cells, and then the silencing of a target gene is induced.

[0046] As used herein, the term "antisense" is intended to refer to an
oligomer having a sequence of nucleotide bases and a subunit-to-subunit
backbone that allows the antisense oligomer to hybridize with a target
sequence in RNA by Watson-Crick base pairing to form an RNA:oligomer
heteroduplex within the target sequence, typically with mRNA. The
oligomer may have exact sequence complementarity to the target sequence,
or near complementarity thereto. These antisense oligomers may block or
inhibit the translation of the mRNA, and/or modify the processing of mRNA
to produce a splice variant of the mRNA. Thus, the antisense oligomer of
the present invention is an antisense oligomer complementary to a
polynucleotide coding for the eIF3m polypeptide. For gene therapy, the
antisense oligonucleotide according to the presence invention may be
administered by a typical method. The administration of the composition
may lead to preventing or suppressing oncogene expression. For instance,
an antisense oligodeoxynucleotide is loaded onto a microparticle carrier
based on poly-L-lysine by electrostatic attraction as described in J. S.
kim et al., J controlled Release 53, 175-182 (1998) and the
oligonucleotide-loaded microparticle is injected intravenously, but the
present invention is not limited to this method.

[0047] Preferably, the composition according to the present invention may
include a known therapeutic agent, which is directly or indirectly
conjugated to the agent or is present in an unconjugated form. The
therapeutic agent capable of binding to the antibody includes, but is not
limited to, radionuclides, drugs, lymphokines, toxins and bispecific
antibodies. As long as it can exert therapeutic effects on cancer when
conjugated to an antibody or administered in combination with an siRNA,
an shRNA or an antisense oligonucleotide, any known therapeutic agent can
be used in the present invention.

[0048] Examples of the radionuclides include, but are not limited to,
3H,14C,32P,35S,36Cl,51Cr,
57Co,58Co,.sup.59Fe,90Y,125I,.sup.131I, and
186Re.

[0050] eIF3m-specific siRNAs are examined for activity of inhibiting
oncogenesis by suppressing eIF3m gene or protein expression. Comparison
with a control indicated that expression patterns relevant to cell growth
and cell cycle were regulated.

[0051] In a preferred embodiment thereof, the present invention provides a
composition comprising a substance inhibiting the activity of the eIF3m
protein. Preferably, the activity-inhibiting substance is an antibody
that specifically recognizes an eIF3m protein. The antibody includes all
monoclonal antibodies and chimeric antibodies, humanized antibodies and
human antibodies thereof. As long as they have the binding property of
specifically recognizing eIF3m, the antibodies include complete forms
having two full-length light chains and two full-length heavy chains, or
may be in the form of functional fragments of antibody molecules. As used
herein, the term "functional fragments of antibody molecules" is intended
to refer to fragments retaining at least an antigen-binding function,
which are exemplified by Fab, F(ab'), F(ab)2 and Fv.

[0052] Preferably, the composition according to the present invention may
include an acceptable carrier appropriate to the administration mode
thereof.

[0053] The active ingredient may be combined with pharmaceutically
acceptable vehicles, excipients, or additives. Examples of the
pharmaceutically acceptable carriers useful in the present invention
include physiological saline, sterile water, Ringer s solution, buffered
saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and
liposomes. They may be used alone or in combination. If necessary, the
composition may further comprise other typical additives such as
antioxidants, buffers, etc. Depending on administration mode, the
composition may be formulated with a diluent, a dispersant, a surfactant,
a binder and/or a lubricant into an injection dosage form such as aqueous
solution, suspension, emulsion, etc. or an oral dosage form such as pill,
capsule, granule, tablet, etc. When conjugated with the carrier, an
antibody or ligand specific for target organs or tissues may direct the
active ingredient toward the organs or tissues. Typical vehicles,
excipients and additives known in the art may be used in the present
invention. The present invention is not limited to the examples of
vehicles, excipients and additives.

[0054] The composition or formulation may be administered in a
therapeutically effective amount to subjects through a suitable route
according to purpose or necessity. The pharmaceutical composition may be
administered orally, parenterally, subcutaneously, intraperitoneally, or
intranasally. For local immunosuppressive therapy, the composition may,
if desired, be administered using a suitable method, including
intralesional administration. Parenteral injections include
intramuscular, intravenous, intraarterial, intraperitoneal and
subcutaneous routes. The therapeutically effective amount of the
composition comprising the antisense oligonucleotide, siRNA or shRNA may
vary depending on various factors well known in the medical art,
including the kind and degree of the response to be achieved, the
patient's age, body weight, and state of health, etc.

[0055] In accordance with still yet another aspect thereof, the present
invention is directed to a method for screening a curative agent for
cancer, comprising treating a cell expressing an eIF3m polypeptide and/or
polynucleotide with a candidate compound and measuring an eIF3m
polypeptide or polynucleotide expression level in the cell.

[0056] In the screening method of the present invention, the candidate
compound, if inducing an increase in eIF3m expression level, is
determined as being oncogenic. When the eIF3m expression level is reduced
thereby, the candidate compound is determined as a possible therapeutic
agent for cancer. According to the screening method, the activity of the
candidate can be easily determined by the eIF3m expression level.

Advantageous Effects of Invention

[0057] As described hitherto, eIF3m can be used as a cancer marker which
allows cancer to be diagnosed with high sensitivity and specificity.
Particularly, the cancer marker is useful for the diagnosis of lung
cancer, breast cancer, liver cancer, leukemia, lymphoma, rectal cancer,
melanoma, and colon cancer. Furthermore, when administered to a subject,
an eIF3m expression regulator can prevent the onset or progress of cancer
by inhibiting the overexpression of the eIF3m gene.

BRIEF DESCRIPTION OF DRAWINGS

[0058] FIG. 1 shows expression levels of eIF3 subunits in paired patient
tissues. All the qRT-PCR reactions were carried out using primers listed
on Table 1 and quantified by AQ method.

[0066] FIG. 9 shows eIF3m and RNA in immunoprecipitate of HCT-116 cells.
(a) Western blot of eIF3m from the immunoprecipitated cell lysate. Blank
vector control(pFLAG-CMV2) did not show any detectable band in either
input control(IC) or in immunoprecipitate of anti-FLAG antibody
conjugated affinity gel (IP). However, in IP of pFLAG-CMV2-eIF3m
transfected HCT-116 cells the band appeared with designated size. (b) The
RNA resolved on formaldehyde gel after extraction from the
immunoprecipitate. The blank vector control did not give RNA, but in
pFLAG-CMV2-eIF3m transfected cells appeared RNA.

[0069] FIG. 12 shows the specificity of reaction confirmed by sequencing
of PCR product.mRNA levels of MIF (a) and MT2A (b) as measured by qRT-PCR
(relative quantification by normalized with beta-actin mRNA expression)
in HCT-116 cells at various times after eIF3m siRNA-1 transfection.

[0071] FIG. 14 shows that Sub-G0/G1 population of HCT-116 colon cancer
cell line increases when eIF3m expression is silenced. The nuclear
contents of siRNA transfected cells were measured every 24 hours after
treatment by flow cytometry to see the cell cycle progression. BF and NC
controls did not show any significant cell cycle differences among
mitotic cellcycle stages. The eIF3m siRNA-1 treatment increased sub-G0/G1
stage at 24 hours, and remained elevated until 96 hours.

[0072] FIG. 15 shows that the nuclear contents of siRNA transfected cells
were measured every 24 hours after treatment, by flow cytometry to see
the cell cycle progression. There were no significant differences in sub
GO/G1 phase in BF and NC controls through the culture period, but
sub-G0/G1 stage after eIF3m siRNA-3 treatment was elevated until 96
hours.

MODE FOR THE INVENTION

[0073] A better understanding of the present invention may be obtained
through the following examples which are set forth to illustrate, but are
not to be construed as limiting the present invention.

EXAMPLE 1

Colon Tissue

[0074] Human colon tissues were obtained in compliance with the Helsinki
Treaty. Tissues were excised from patients according to the protocol and
stored at -80° C. until use.

EXAMPLE 2

Cell Culture

[0075] All of the cell lines used in the present invention were obtained
from the ATCC (American Type Culture Collection, Manassas, Va.) and the
Korean Cell Line Bank (Seoul, Korea). Each cell line was maintained in a
maintenance medium supplemented with 10% FBS (fetal bovine serum,
Invitrogen).

[0079] Tumor tissues of the colon were fixed in a 10% neutral buffered
formalin solution and embedded in paraffin blocks according to the
standard procedure. 4 μm-thick tissue slices were mounted on slides
and were pretreated with proteinase. Staining was conducted using a
BechMark XT automated system (Ventana Medical System). A primary antibody
for Western blotting was applied at 1:100 dilution and masked with
endogenous biotin to avoid the detection of false positive signal.
Signals were detected by biotinylated secondary antibodies, followed by
binding a streptavidin-HRP (horseradish peroxidase) conjugate thereto.
The complex was visualized as dark brown precipitates with a hydrogen
peroxide substrate and 3,3'-DAB (diaminobenzidine tetrahydrochloride)
chromogen The slides were counterstained with hematoxylin-eosin and
observed under an optical microscope.

EXAMPLE 7

Cloning of Tagged eIF3m

[0080] The eIF3m ORF was amplified with primers having the following
nucleotide sequences and inserted into a pCR2.1-TOPO vector.

[0081] After being sequenced, the insert was cloned into a pFLAG-CMV2
vector (Sigma-Aldrich).

EXAMPLE 8

Ribonomics

[0082] Two million HCT-116 cells were transfected with 4 μg of the
pFLAG-CMV2-eIF3m expression vector by Lipofectamine 2000 (Invitrogen) and
cultured for 48 hrs at 37° C. in DMEM supplemented with 10% FBS in
a 5% CO2incubator. The cells were homogenized in Symplekin
immunoprecipitation buffer (150 mM NaCl, 25 mM HEPES-KOH [pH 7.5], 10%
[v/v] glycerol, 1 mM MgCl2, 2 mM sodium orthovanadate, 2 mM
β-glycerophosphate, 1 mM PMSF (phenylmethylsulphonylfluoride), 1 mM
DTT, 2 mM EDTA, 0.5% TritonX-100, 50 μg/ml RNaseA [Sigma-Aldrich], and
1× protease inhibitor cocktail [Roche]). The lysate was washed,
centrifuged, and precipitated with 40 μl of FLAG-M2 affinity gel.
Total RNA was extracted from immunoprecipitated gel pellet using Trizol
reagent according to the protocol provided by the manufacturer. The total
RNA was quantified with Nanodrop 1000. 1.575 μg of total RNA was used
for making cDNA library by GeneRacer kit (Invitrogen). The total RNA was
dephosphorylated, decapped and ligated to an RNA adaptor before reverse
transcription. Using GeneRacer 5' and 3' primers specific for the
adaptor, PCR was performed with 24 cycles of the following thermal
profiles. The PCR product was cloned into an EcoRV-digested
pBlueScriptll-KS(+) vector and transformed into DH10B competent cells by
electroporation. Cells from single colonies were identified and each
clone was sequenced.

EXAMPLE 9

Cell Proliferation and Cell Cycle Analysis

[0083] The effects of eIF3m on cell proliferation and cell cycle were
assayed after the treatment of the wild-type human colon cancer cell line
HCT-116 with siRNAs which were designed in advance from Qiagen. 20,000
HCT-116 cells were transfected with eIF3m-specific siRNA, or negative
control siRNA (NC) or RNase-free buffer only (BF) and cultured for 96
hours in 6-well plates, with monitoring for cell proliferation by MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay
every 24 hours. The three kinds of HCT-116 cell cultures with
non-specific control siRNA, eIF3m-specific siRNA and RNase-free buffer
only (untreated 100% survival control) were transected and plated onto
6-well plates. 725 μL of MTT (2 mg/ml) was added to each well every
hour. After incubation for 4 hrs, the medium was aspirated off and the
cells were incubated for 10 min with 1.955 ml of DMSO (dimethyl
sulfoxide). Absorbance at 540 nm was read on a scanning microplate reader
(Molecular Devices). Cell cycle progression was analyzed through flow
cytometry. Approximately 5×104 HCT-116 cells were seeded onto
6-well plates and treated with negative control siRNA, eIF3m-specific
siRNA or RNase-free buffer only. Cells were collected by treatment with
0.5% trypsin every hour, washed with ice-cold PBS and fixed at 4°
C. with 70% ethanol. Then, the cells were washed and resuspended at
37° C. for 20 min in propidium iodide (PI) staining buffer (10
lag/m1DNase-free RNase A, 50 μg/ml PI in PBS). The PT-stained
cells were analyzed using BD Cellquest software on FAC-SCalibur® Flow
Cytometer (BD Biosciences, USA). eIF3m expression was found to be reduced
(knockdown), as monitored by Western blotting.

EXAMPLE 10

Assessment of Statistical Significance

[0084] The statistical significance was analyzed by Student's two-tail
t-test for comparison between unpaired groups and Student's paired
two-tail t-test for comparison between normal/tumor tissue pairs. For
displaying error range we adopt standard deviation or standard error mean
(SEM) for each group

Results

[0085] Elevated Expression of eIF3m Subunits in Cancer

[0086] To examine relationship between the upregulation of eIF3m subunit
and the progression of human colon tumor, real-time PCR was first
performed to detect transcripts of 13 eIF3 subunits in pairs of normal
colon and tumor (adenocarcinoma) tissues from 19 patients. Expression
differences were measured by relative quantification (RQ) normalized by
GAPDH (FIG. 1). eIF3b, one of the eIF3 core subunits, showed high
expression levels in most of the patients (18/19) whereas the other core
factors remained low in expression level. Among the eight non-core
subunits, eIF3d, e, h, k and in subunits showed elevated expression in
tumors of more than 50% of patients. Of the five non-core eIF3 subunits
showing higher expression in tumors, eIF3m was chosen for further study
on characteristics thereof. In order to confirm the elevated eIF3m
expression in tumors, all the same patients as well as seven kinds of
cultured cancer cell lines were measured for eIF3m mRNA levels by
absolute quantification (AQ). A standard curve (correlation coefficient
r2>0.95) was drawn, and used to calculate the numbers of molecule
(FIG. 2A). The lung cancer cell line, A549, showed 4.25×104
copies/ng total RNA. In four colon cancer cell lines, the expression
ranged from 1.92×104 to 3.44×10 copiesing total RNA. In
two breast cancer cell lines, especially in Herceptin resistant JIMT-1,
the expression level (6.09×104 copies/ng total RNA) was more
than double of the Herceptin sensitive SK-BR-3(2.57×104
copies/ngtotalRNA). In contrast to those seven human cancer cell lines,
the normal cell line HDF showed a very low expression level
(1.17×103 copies/ng total RNA). While the RQ data showed
higher expression in tumors in 12 out of 19 patients, AQ showed markedly
elevated expression of eIF3m in tumors in all patients (paired t-test
p=0.00013) ranging from 3.44×102 to 3.27×104
copies/ng total RNA compared to the normal counterparts which ranged from
3.50×10 to 2.51×103 (FIG. 2B). Also, elevated protein
levels of eIF3m were confirmed in the cell lines and tissue pairs. Cancer
cell lines expressed higher protein levels of eIF3m than did normal cell
line HDF, with the correspondence of their expression intensities with
mRNA levels (FIG. 2C). The eIF3m expression in two breast cancer cell
lines at protein level was similar to the mRNA levels described above.
There is a difference in eIF3m protein expression between normal and
tumorous colon tissue, which was however not as dramatic as in the mRNA
levels (FIG. 2D).

[0087] To make sure of its expression difference, we checked its mRNA and
protein levels (FIGS. 3A and 3B) in 20 additional paired patient tissues.
Then protein expression difference was evaluated between normal and tumor
tissues by densitometry (FIG. 3C). The average density of eIF3m itself in
tumor tissues (86.6) was 1.3 fold higher than in normal counterpart
(66.6) (paired t-test p=0.00098). The fold change of normalized density
by beta-actin was also 1.3 (p=0.00070).

[0088] Data obtained from the analysis imply that the elevated expression
level of eIF3m is associated with tumor progression in cell lines as well
as in colon tissues.

[0089] Tissue Specificity of eIF3m mRNA Expression

[0090] The tumor specificity of eIF3m expression was examined at a
transcription level by Northern hybridization. While the normal tissues,
heart, skeletal muscle, kidney, liver, placenta, and peripheral blood
leukocytes showed elevated eIF3m expression, no detectable expression
signals of eIF3m were found in brain, colon, thymus, small intestine, and
lung. β-actin, serving as a housekeeping gene, was consistently
present in all these tissues (FIG. 4A). Human cancer cell lines also
showed high expression of eIF3m mRNA (FIG. 4B). Very high expression
levels of eIF3m were observed in several leukemia cell lines and lymphoma
cell lines. The colorectal adenocarcinoma SW480 was higher in eIF3m
expression than was melanoma. Also, the lung carcinoma cell line A549 was
found to have an eIF3m expression level as measured by qRT-PCR. In this
hybridization no splice variants of the eIF3m transcript was found.

[0091] Normal/tumor tissue pairs from colorectal adenocarcinoma patients
were assayed for eIF3m expression at a transcription level by qRT-PCR
(FIG. 5). Tumor tissues from most of the patients were observed to
significantly increase in eIF3m mRNA level, with approximately 10-fold
higher expression levels than in normal tissues in some patients (FIG. 5A
#33 and #34). In a Western blotting analysis, significantly thick bands
were detected compared to those of normal tissues. (FIG. 5B)

[0092] Confirmation of the Preferential Expression of eIF3m in Human Colon
Carcinoma Tissues

[0093] The expression of eIF3m was also detected as measured by qRT-PCR
and Western blotting. To examine whether eIF3m is expressed only in
particular cells of tissues, immunohistochemistry (IHC) was performed on
tissue sections. A primary antibody for use in Western blotting was
applied to well-differentiated lymph nodal metastatic colorectal
adenocarcinoma cells and moderately differentiated colorectal
adenocarcinoma cells (FIGS. 6D and 6F). As a result, a remarkably high
expression level of eIF3m was detected in cells within tumor regions
(FIGS. 6C and 6F) and in the metastatic carcinoma cells in regional lymph
node (LN) (FIG. 6A). In contrast, low signals were detected in
non-neoplastic epithelial cells of peritumoral tissues which are located
in the lining lumen of the colon where cellular regeneration occurs
constantly (FIGS. 6B and 6E). The subcellular localization of eIF3m
expression was confirmed at higher magnification. eIF3m expression was
not detected in the blue counterstained nuclei but was confined to the
cytosol in both carcinoma and non-neoplastic epithelial cells. Compared
to epithelial cells in crypt regions, the matrix cells present
therebetween had lower expression levels. No eIF3m was observed in the
muscular cells (FIGS. 6A and 6D). A high expression level of eIF3m was
observed in metastatic carcinoma on hepatic tissues (FIGS. 6G to 6I).
Whereas peritumoral tissues were relatively clear, metastatic regions
showed a strong expression of eIF3m in a pattern similar to that in
tumors of colorectal adenocarcinoma, as shown in FIG. 6G and 6F. A high
eIF3m expression level was also detected in hepatocellular carcinoma
(FIGS. 6J and 6L). RN (regenerating nodules) in cirrhosis regions
characterized by responsive proliferation also showed strong expression
of eIF3m, compared to peripheral region of normal tissues. Similarly,
serous cystadnocarcinoma showed high expression levels of eIF3m than did
peritumoral regions of the ovarian tissues (FIGS. 6M and 6O).

[0094] These results suggest that eIF3m is highly expressed in tumor and
peritumoral tissues where cell proliferation is active at higher level.

[0096] The low expression of eIF3m in the peritumoral regions confirmed by
IHC, suggests that eIF3m is also involved in reactive proliferation as
well as in tumor progression. To determine the effect of eIF3m expression
on cell proliferation, we studied the effect of silencing eIF3m mRNA in
HCT-116 colon cancer cells using siRNA. First, we transfected HCT-116
colon cancer cells with buffer (BF), non-human negative control siRNA
(NC), and eIF3m specific siRNA-1/-3. Then we examined the expression of
eIF3m every 24 hours for 96 hrs after transfection by Western blotting
(FIGS. 7A and 8A). eIF3m expression was reduced from 24 hrs after siRNA
transfection until 96 hrs. In contrast, BF or NC did not change the level
of eIF3m protein. The confluency after BF and NC rapidly increased and
reached a plateau at 72 hrs (FIG. 7B), but eIF3m siRNA-1 slowed down the
proliferation from 24 hrs. The confluency at 96 hrs of eIF3m siRNA was
lower than that with BF or NC (FIG. 7B). MTT assays confirmed this by
showing no more increase than 3 fold with eIF3m siRNA at 72 hrs compared
to 6 fold with NC and 5 fold with BF (FIG. 7C). This result was confirmed
again by using another siRNA, siRNA-3 (FIG. 8B). Silencing efficacies of
siRNA-1 and -3 compared to NC at 96 hrs were 46.4% (Student's t-test
p=1.3×10-5) and 65.3% (Student's t-test
p=2.8×10-9), respectively. These results suggest that
proliferation of cancer cells is retarded by silencing eIF3m expression.

[0097] eIF3m-Associated Transcript

[0098] eIF3m is known as one of the "non-core" eIF3 subunits. Putative
eIF3m is one of the components of the 40S ribosomal subunit and can be
involved in the expression of a subset of transcripts in association with
cell proliferation depending on physiological state of the cell. Thus,
the present inventors employed a ribonomics strategy to find transcripts
which were expressed at a high level in association with eIF3m. eIF3m was
cloned into a FLAG tagging expression plasmid and expressed under the
control of CMV2 promoter in HCT-116 colon cancer cell lines. After 48
hours of transfection, the cultured cells were harvested and the lysate
was immunoprecipitated with FLAG-M2 affinity gel. The eIF3m-inserted
plasmid generated a designated size of eIF3m band on Western blots, but
the blank vector control did not (FIG. 9A). Total RNA was extracted from
the immunoprecipitated gel pellet. The eIF3m expression clone generated a
detectable amount of RNA in the immunoprecipitate but a blank vector did
not (FIG. 9B). This RNA was used to make a cDNA library. Among the 344
randomly picked clones, sequences of 181 clones were successfully read
and 81 representative kinds of eIF3m-associated genes are given in Table
2, below. These sequences include 75 ribosomal protein genes (41.4%),
eIF3m itself (25.4%), 54 representative single genes (29.8%), and 8
unclassified entries (4.4%). Classification of these genes by gene
ontology for molecular function revealed that most of the
eIF3m-associated transcripts encode genes for protein translation
(protein binding, structural constituent of ribosome, translation
initiation factor activity, and ribonucleoprotein binding) and RNA
binding (nucleic acid binding, RNA binding, and rRNA binding) (Table 2).
Another notable class of genes was concerned with metal ion binding
activity (cadmium ion and copper ion binding). For an advanced study on
these transcripts, a choice was made of macrophage migration inhibitory
factor (MIF)gene. When eIF3m siRNA-treated HCT-116 cell lysate was
examined by Western blot, the protein level of MIF decreased until 48
hours as was eIF3m expression but it revived to normal level from 72 hrs
(FIG. 11A). UCIMT antibody detects both MT1 and MT2 isoforms, but MT2A
was major form in ribonomics, so we applied the antibody to detect MT2A
protein. The protein level of MT2A also decreased until 48 hrs, but did
not return back to normal level (FIG. 11B) unlike MIF. However, mRNA
level did not correlate with protein level but showed minor change in
different way (FIGS. 11A, 11B and 11C)). This suggests that eIF3m
influence on the expression of a designated subset of cell proliferation
genes by modulating translation efficiency or affecting stability of
target mRNA.

[0099] Also, the protein level of MIF and MT2A was examined for whether it
is influenced by eIF3m expression in the HCT-116 cells. Since, Lim et al.
(2009) reported cell division cycle 25 homolog A (CDC25A) degradation and
G1-arrest by MT2A silencing in breast cancer cells, whether eIF3m
silencing affects CDC25A level was checked and it was immediately found
that it decreased CDC25A from 24 hrs until 72 hrs (FIG. 13). These
results confirm eIF3m expression influence on cell cycle regulation.

[0100] Table 2

[0101] Effect of eIF3m Silencing on Cell Cycle

[0102] Cell proliferation or cell death is closely associated with cell
differentiation since a continuous cell cycle is the first entrance of
determining both proliferation and apoptosis. Hence, in the context of
the cell proliferation retardation, as shown in FIG. 7, by eIF3m
silencing, the cell cycle progression following eIF3m knockdown in
HCT-116 cell lines was analyzed using flow cytometry. The HCT-116 cells
were treated with eIF3m siRNA, BF, or NC controls. After 24 hrs of siRNA
transfection, there was no significant difference in the proportion of
each mitosis stage between groups treated with NC, BF, and eIF3m siRNA
(FIGS. 14 and 15). As the time point moves from 24 hrs to 48 hrs, the
portion of sub-G0/G1 phase in eIF3m siRNA-treated cells increased from
1.65% to 7.87%, but sub-G0/G1 of BF and NC remained at 1.44% and 2.37%,
respectively. Interestingly, the proportion of S phase of eIF3m siRNA-1
treated cells decreased to 22.16% from 26.40% while it increased from
24.67% to 30.12% and from 26.00% to 31.06%, respectively in BF and NC.
When the cells were cultured for 72 hrs, the increment of sub-G0/G1
portion of eIF3m siRNA-1 (11.36%) became more prominent compared to BF
(1.97%) or NC (2.78%). The S phase in the case of eIF3m siRNA-1 at the
same time point (23.73%) was still lower than those with BF (29.15%) or
NC (25.67%). In addition, the G2/M phase that did not show significant
difference at previous time points decreased to 22.91% compared to 27.03%
with BF and 29.46% with NC at 72 hrs. This trend was maintained for 96
hrs, sub-G0/G 1 phase reaching 12.36% in eIF3m siRNA-1 while remaining
low in BF (0.49%) and NC (0.91%). On the other hand, the S phase became
much lower in eIF3m siRNA-1 (19.49%) compared to BF (32.22%) and NC
(30.56%), and similarly decreased in G2/M phase (21.81%) compared to BF
(25.63%) and NC (27.40%). eIF3m siRNA-3 also showed increased sub GO/G1
from 24 hrs and at 96 hrs it showed 11.26% of sub GO/G1 compared to BF
(2.52%) or NC (3.31%) and decreased G2/M-phase (19.75%) portion compared
to BF (31.16%) or NC (29.22%) . Taken together, these results suggest
that silencing of eIF3m expression increased sub-GO/G1 proportion and led
to cell death. Thus, eIF3m expression seems to be required for the cells
to continue cell-cycle and eventually for cell proliferation.

[0103] Although the preferred embodiments of the present invention have
been disclosed for illustrative purposes, those skilled in the art will
appreciate that various modifications, additions and substitutions are
possible, without departing from the scope and spirit of the invention as
disclosed in the accompanying claims.